The long term goal of this Program is to develop gene transfer methods for the treatment of neural disorders. Three groups that are well integrated have come together to develop methods for using recombinant Adeno-associated virus (rAAV) for the treatment of retinal and CNS neurodegenerative diseases. Project 1 (Muzyczka) proposes genetic experiments to identify the proteins in the substantia nigra and striatum that interact with ct synuclein. It will specifically examine a-syn interactions with GRK and PLD2, and develop for the first time somatic knockouts of GRK and PLD2 using AAV vectors. It will also examine the effect of oxidative stress in combination with overexpression on neurodegeneration in the substantia nigra. Finally, it will use biochemical techniques to directly identify protein complexes that contain a-syn. Project 2 (Hauswirth and Lewin) will take the next step toward developing a therapy for P23H rhodopsin RP using the ribozymes they developed in the previous grant period. Further, they will test two new strategies for RP that are likely to be of more general use for all RP diseases. The first is the use of GDNF expression to promote photoreceptor survival. The second is to replace all (wild type and mutant) endogenous rhodopsin mRNAs with a wild type mRNA. If successful, this should prove to be a general approach that could be applied to all genetic RP, regardless of the point mutant involved. Project 3 (Mandel) will extend their preclinical experiments toward developing AAV mediated gene transfer for Parkinson disease. Specifically, they will develop regulatable GDNF constructs that are a prerequisite for clinical applications, do the first comprehensive analysis of the immune response to AAV vectors that are injected into the brain, and test their therapeutic GNDF strategy in a primate model of Parkinson's to obtain dosing information and confirmation of efficacy in a brain model closer to human. Two cores are also proposed. Core A (Administration) will continue in its role of providing fiscal/administrative support, educational programs, and program oversight in the form of internal and external advisors. Core B (Vector Core) will continue to improve the efficiency and scaleability of rAAV vectors. In addition to providing the routine service of production and purification of rAAV2-based vectors, the Core will also develop methods for purification of alternative AAV serotypes and capsid mutants to be used in projects 1, 2, and 3. [unreadable] [unreadable] PROJECT 1 [unreadable] [unreadable] Principal Investigator: Muzyczka, Nicholas [unreadable] [unreadable] Title: a-Synuclein Function [unreadable] [unreadable] Description (provided by applicant): Recent data from our lab and others have demonstrated that targeted a-synuclein overexpression in the substantia nigra pars compacta leads to Parkinson-like neurodegeneration. Yet, the function of a-synuclein is still unknown. The present proposal attempts to define the function of a synuclein in both normal brain function and in the pathogenesis of Parkinson disease (PD). a-synuelein is likely to be part of a multiprotein complex, and understanding the interaction between a-synuclein and its protein binding partners should help in understanding the mechanism underlying pathogenesis in PD as well as its function in the normal brain. It might also help explain the selective vulnerability of certain neuronal populations to a-synuclein. We propose to use recombinant Adeno-Associated Virus (rAAV) as a gene delivery vector to generate regionally specific somatic transgenics of the nigrostriatal tract. The following 3 aims are proposed. The first aim is to examine the effect of down regulating or overexpressing genes that are known to interact with a-synuclein, Phospholipase D2 (PLD2) and/or G coupled Receptor protein Kinase 2 (GRK2) will be overexpressed or knocked out in combination with a-synuclein to determine whether the known interactions between these proteins and a-synuclein are involved in the pathogenesis of PD, We will follow the progression of neurodegeneration caused by these combinations by looking at the number of TH- positive neurons and behavioral deficits. Aim 2 is to identify additional proteins that interact directly with a synuclein. Tagged rat and human synuclein will be used as a bait to affinity-immuaoprecipitate a synuclein-protein complexes in vivo in the dopaminergic MN9D cell line. The affinity purified complexes will be analyzed by mass spectrometry methods and antibody to known interaction partners to identify the multiprotein interactions for a-synuclein. Aim 3 is to determine whether the toxicity of dopamine related oxidative stress is a factor in the selective vulnerability of certain dopaminergic neurons to Parkinson-like neurodegeneration induced by a-synuclein. AAV will be used to deliver the a-synuclein gene alone, or in combination with genes that increase or decrease oxidative stress in substantia nigra. These include the tyrosine hydroxylase (TH) and GTP cyclohydrolase genes to increase nigrostriatal dopamine production by overexpression of the precursor L-dopa, ribozymes to mitochondrial complex 1 to increase oxidative stress and catalase to reduce oxidative stress. As in aim 1 we will follow the progression of neurodegeneration caused by these combinations. [unreadable] [unreadable]